A new lipase (Alip2) with high potential for enzymatic hydrolysis of the diester diethyladipate to the monoester monoethyladipate

Highlights

• Identification of new lipase Alip2p from the yeast Blastobotrys raffinosifermentans (Arxula adeninivorans).

• Alip2p selectively hydrolyzes symmetric diesters to monoesters.

• Enzymatic synthesis of monoethyl adipate from the diester.

Abstract

Several putative lipase genes from the genome of the yeast Blastobotrys (Arxula) raffinosifermentans (adeninivorans) LS3 were overexpressed in the yeast itself and screened for the desymmetrization of the dicarboxylic acid diester diethyl adipate (DEA) into the monoester monoethyl adipate (MEA). MEA can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications.

The selected lipase Alip2-c6hp was intracellularly located. After overexpression of the corresponding gene, it was purified and biochemically characterized using p-nitrophenyl butyrate as the substrate for standard activity tests. In fed-batch cultivation with constructed yeast strain B. raffinosifermentans G1212/YRC102-Alip2-c6h for large scale production of the Alip2-c6hp biocatalyst enzyme activities up to 674 U L⁻¹ were reached.

Several tested diesters were hydrolyzed selectively to monoesters. Under optimized conditions, the purified enzyme Alip2p-c6h converted 96 % of the substrate DEA to MEA within 30 min incubation, whereby only 1.6 % of the unwanted side-product adipic acid (AA) was formed. At room temperature the dicarboxylic acid esters diethyl malonate (DEM), diethyl succinate (DES), dimethyl adipate (DMA) and dimethyl suberate (DMSub) were completely hydrolyzed to their corresponding monoesters. A high yield of 87 % and 25 % could also be achieved with the dioldiesters 1,4-diacetoxybutane (DAB) and diacetoxyhexane (DAH).

In conclusion the potential of the lipase Alip2-c6hp expressed in B. raffinosifermentans is very promising for selective hydrolysis of DEA to MEA as well as for the production of other monoesters.

Introduction

Monoesters of symmetrical dicarboxylic acids such as monoethyl adipate (MEA) of diethyl adipate (DEA) can serve as a monomeric spacer group for functional polymers used in medical chemistry and dental applications and there is an increased demand for those substances.

Polymers with functional groups, known as functional polymers, are of increasing interest in different areas including sensors, energy storage, food, biomedical and environmental applications [1]. Spacer groups are often an essential part of these polymers. They serve as ‘distance holder’ between polymer material and the functional group to ensure the accessibility and improve the binding ability. The length and type of the respective spacer arm determines chemical, physical and biological properties as well as the flexibility and the formability [2].

In addition, dicarboxylic acid monoesters and diol monoesters are key substances in the pharmaceutical industry or serve as building blocks in the chemical industry [[3], [4], [5]].

Monoesters can be synthesized chemically by (1) acid or base catalyzed hydrolysis of the diesters, (2) esterification of a dicarboxylic acid with an alcohol or diols with carboxylic acids and (3) transesterification of the diester. But these reactions are often not selective: both ester bonds can be cleaved or both carboxyl or hydroxy groups can be (trans)esterified. This is leading to a mixture of the monoester, diester and diacid and thus monoester yields are low depending on the method used. The educt’s structure also affects the yield and selectivity of the reaction products, as it was shown for the microwave assisted hydrolysis of carboxylic esters by InI₃/SiO₂/H₂O yielding between 70 and 93 % depending of the length and structure of the substrate [6].

In contrast to chemical synthesis, enzymatic reactions have many advantages. They can be selective, so that high monoester concentrations can be reached. The affinity and rate for the diester should be much higher than for the monoester to obtain especially monoester as a product.

Enzymatic syntheses for the preparation of dicarboxylic acid monoesters and diol monoesters have already been used successfully with yields up to 100 % [[4], [5], [6], [7], [8]]. In particular, lipases (EC 3.1.1.3) are currently used as biocatalysts for the efficient production of dicarboxylic acid monoesters by the selective hydrolysis of diesters [4,5,[7], [8], [9], [10]].

The yeast Blastobotrys raffinosifermentans (syn. Arxula adeninivorans) LS3 [11] offers a high potential to uncover of new lipases because the yeast can use, among other substrates, a number of lipophilic compounds as the sole source of energy and carbon [12]. The thermo- and halotolerant yeast was already used in the 1980s for ‘single cell protein production’ in Malchin (Mecklenburg-West Pomerania, Germany) because of its excellent growth parameters. In addition, its C and N source spectrums are exceptionally extensive for yeast. Based on these properties, B. raffinosifermentans has been used as a producer of a number of proteins [13], in particular such as Interleukin-6 [14] and Human Serum Albumin [[15], [16], [17]], and enzymes such as the feed additive phosphatase APHO1 with phytase activity [18], tannase [19,20] and the extracellular lipase Alip1p [12].

In this study, the genome of B. raffinosifermentans LS3 was screened for putative lipases with the ability for monoester production. After a selection, the Blastobotrys-endogenous, putative lipase Alip2p was purified, characterized and its potential for the production of the monoester monoethyl adipate (MEA) of the symmetrical dicarboxylic acid diethyl adipate (DEA) by hydrolysis was evaluated.